Calycosin-triblock copolymer nanomicelles attenuate doxorubicin-induced cardiotoxicity through upregulation of ERp57

2.1 Materials

Doxorubicin hydrochloride (Dox), Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), and 2, 7-dichlorodihydrofluorescein diacetate (H2-DCFDA) were purchased from Sigma-Aldrich (St Louis, MO, USA). RPMI-1640 and fetal bovine serum (FBS) was obtained from Gibco BRL (Grand Island, NY, USA).

3.1 Preparation of calycosin- loaded triblock copolymer nanomicelles

Calycosin-loaded PEG-PPG-PEG nanomicelles were prepared via dialysis approach. Briefly, calycosin (0.3 mg/ml) and the PEG-PPG-PEG copolymer (3 mg/ml) were hydrated in 10 ml of dioxane and the solution was mixed and stirred at 500 rpm for 50 min, followed by addition of 3 ml deionized (DI) water drop-wise for 60 min under constant stirring. The obtained micellar solution was then transferred into dialysis membrane (MWCO 700 g/mol) against DI water for 5 h. Empty PEG-PPG-PEG nanomicelles were also synthesized by the similar method in the absence of calycosin.

3.2 Characterization of the calycosin- loaded triblock copolymer nanomicelles

The hydrodynamic size, polydispersity and zeta-potential of prepared nanomicelles were explored using a Zetasizer analyzer (Malvern, UK). Samples were dispersed in DI water and analyzed at room temperature. The morphology of the nanomicelles was determined using transmission electron microscopy (TEM, Philips, CM-100, Germany). A drop of sample was dropped on the copper grid-carbon film and dried at room temperature. The critical micellar concentration (CMC) was determined by using 1,6-diphenyl-1,3,5-hexatriene (DPH) probe, which 25 ml DPH solubilized in methanol (0.5 mM), was mixed with micellar solution with different concentrations ranging from 0.001 to 0.5 mg/mL and the absorbance was read at 350 nm using UV–vis spectrophotometer (Beckman Coulter).

3.3 Encapsulation efficiency and calycosin content determination

Dialysis method (MWCO 700 g/mol) was used to determine the amount of calycosin encapsulation based on using UV–vis spectrophotometry at 428 nm (Beckman Coulter) using a standard calibration curve. The Dox loading capacity (LC) was determined as a difference between the initial concentration of calycosin and the concentration in the aqueous sample after the dialysis method. The encapsulation efficiency (EE) was calculated based on the following equation (Li et al., 2017): $$\mathrm{E}\mathrm{E}(\%)=(\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}-\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n})/\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}.$$

3.4 In vitro release study

In vitro release of calycosin from the PEG-PPG-PEG nanomicelles was investigated in a PBS buffer (pH 7.4, 100 mM) at room temperature. The prepared micellar solution was poured into a dialysis bag (MW = 7000 g/mol) and immersed into 200 ml of PBS containing 2% ethanol under constant stirring (50 rpm) at 37 °C. At different time intervals, a defined amount of sample was withdrawn and replaced by fresh PBS buffer and the concentration of the released calycosin was quantified by UV–vis spectrophotometry as stated above.

3.5 Antioxidant activity of free and calycosin- loaded triblock copolymer nanomicelles

The antioxidant activity of free and micellar calycosin with a concentration of 0.1 mg/mL was explored by ABTS assay kit (CS0790, sigma, USA) and hypochlorite assay kit (2687100, Hach Co. USA) based on the manufacturer's protocols. The antioxidant activity of samples was estimated using the following equation:

Antioxidant activity = (A_o- A)/A_o, where A_o is the absorbance of the control and A is the absorbance of samples containing free calycosin or calycosin-loaded nanomicelles. Vitamin C with a concentration of 1 mg/mL was used as a positive control.

3.6 Cell culture

Cardiac muscle cells (H9C2 cells) were cultured in RPMI-1640 cell culture medium supplemented with FBS 10% (v/v) and 1% penicillin-streptomycin in a humidified atmosphere at 37 °C with 5% CO₂.

3.7 MTT assay

The cytotoxicity of the Dox, free calycosin, calycosin- loaded triblock copolymer nanomicelles, and empty triblock copolymer nanomicelles with different Dox or calycosin concentrations (0.01, 0.1, 1, 10, 20, 50 μg/mL) against H9C2 cells was determined by MTT assay. After 24 h incubation, 10 μL of MTT solution was added to each well and incubated for another 4 h, and then 200 μL of DMSO was added to the samples. The absorbance was then read at 570 nm using a microplate reader (BioRad, USA).

3.8 Measurement of intracellular ROS generation

The DCFH-DA assay was used (Alarifi et al., 2017) to determine the antioxidant effects of free calycosin or calycosin- loaded triblock copolymer nanomicelles against Dox-induced ROS production. The cells were exposed to a IC₅₀ concentration of Dox (a concentration of Dox inhibit the viability of cells by 50%), Dox and calycosin (10 µg/ml, the maximum dose with no cytotoxicity), Dox and calycosin- loaded triblock copolymer nanomicelles (10 µg/ml calcysocin), and Dox and triblock copolymer nanomicelles (equivalent concentration to 10 µg/ml calycosin). After incubation for 24 h, the cells were incubated with 10 μM DCFH-DA for 30 min and the fluorescence intensity of the samples was read using a fluorescence microplate reader (BioRad, USA) and the relevant images were taken by fluorescence microscopy (Zeiss, Germany).

3.9 Intracellular RNS measurement

The RNS level was assessed employing RNS Assay Kit (BestBio, China). Cells were washed, stained with BBoxiProbeTM R21F (10 µM) for 30 min, and washed aganin with PBS. Finally, RNS level was determined by using a fluorescence microplate reader (BioRad, USA).

3.10 Silencing ERp57 mRNA

ERp57-siRNA (5′-GGGCAAG GACUUACUUAUU-3′) was employed to silence the ERp57 mRNA based on a previous report (Li et al., 2019). Also, a random nucleotide sequence (5′-UUCUCCGAACGUGUCACGU-3′) was employed as a negative control (NC) siRNA. Transfection of siRNA (50 nM) was done using Lipofectamine 2000 (Invitrogen, USA) based on the manufacturer's instruction and incubated for 48 h. Total mRNAs and proteins of the cells were then extracted and used for quantitative polymerase chain reaction (qPCR) and western blot assays, respectively.

3.11 qPCR assay

After treatment, the cells were collected, washed, total RNA was extracted (TRIzol reagent, Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using a PrimeScript RT Reagent kit (Takara Bio, Japan). qPCR was carried out based on SYBR Premix Ex Taq II (Takara Bio, Japan) based on the manufacturer's instructions. Table 1 shows the primers were used in the present study. The qPCR assay was done with an ABI system (PRISM 7500; Thermo Fisher Scientific, Inc.) based on the 2^-ΔΔCt method (Schmittgen and Livak, 2008).

3.12 Western blot analysis

The cells were transfected with ERp57-siRNA for 48 h, followed by addition of a single concentration of Dox (IC50 concentration of Dox), Dox and calycosin (10 µg/ml, the maximum concentration with no cytotoxicity), Dox and calycosin- loaded triblock copolymer nanomicelles (10 µg/ml calcysocin), and Dox and triblock copolymer nanomicelles (equivalent concentration to 5 µg/ml calycosin). Afterward, the western blot analysis was done based on the previous report (Huang et al., 2020). The following antibodies were used: Monoclonal anti-ERp57 (cat. no. sc80648, Santa Cruz, CA, USA), monoclonal anti-p53 (cat. no. WL01804, Wanlei Biotechnology, Shenyang, China), and monoclonal anti-GAPDH (cat. no.60004-1-lg, ProteinTech Group Inc. Wuhan, China) and diluted to 1:1000 (Huang et al., 2020). The secondary antibodies (anti-mouse cat. no. ZB-2305; ZSGB-Bio, Beijing, China) were diluted to 1:10,000 and the signal was detected using a chemiluminescence kit (cat. no. WLA003a; Wanlei Life Science, Shenyang, China) using a chemiluminescence detection system (Tanon-5200, Shanghai, China).

3.13 Statistical analysis

Student t-test was used to compare two sets of data and one-way analysis of variance followed by a post-hoc Tukey's test was used to compare multiple groups using SPSS version 22.0 (IBM SPSS, Armonk, NY, USA). Data are presented as means ± standard deviation (SD) of three independent experiments. P < 0.05 was considered to show a statistically significant difference.

4.1 Characterization of nanomicelle

DLS analysis for free PEG-PPG-PEG nanomicelles and calycosin- PEG-PPG-PEG nanomicelles showed hydrodynamic radii of about 73 ± 6.11 nm (PDI: 0.241) and 83.27 ± 7.21 (PDI: 0.268), respectively (Fig. 1A). It was observed a monomodal particle size distribution for both empty and loaded-nanomicelles with a slight difference in the size distribution. TEM image also demonstrated that the size of the spherical-shaped calycosin- PEG-PPG-PEG nanomicelles is about 20–30 nm (Fig. 1A, insert).

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Fig. 1

(A) Particle size distribution of free and calycosin-PEG-PPG-PEG nanomicelles. The inset shows the TEM image of nanomicelles. (B) Zeta potential values of free and calycosin-PEG-PPG-PEG nanomicelles. (C) FTIR spectra of calycosin, PEG-PPG-PEG copolymer and calycosin-PEG-PPG-PEG compound. (D) CMC determination based on UV/vis spectroscopy. Data are presented as means ± (SD) of three independent experiments.

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This data was also confirmed with the outcomes determined by zeta potential analysis (Fig. 1B). These data clearly showed that the zeta potential value of free PEG-PPG-PEG nanomicelles was about –21.9 ± 3.98 mV, which decreased to −17.8 ± 2.68 mV upon encapsulation of calycosin (Fig. 1B), indicating the successful loading of drugs into the nanomicelles.

To further assess the potential fabrication of calycosin-PEG-PPG-PEG, FTIR analysis was carried out. The FTIR spectra of calycosin, PEG-PPG-PEG and calycosin-PEG-PPG-PEG are shown in Fig. 1C. The peaks are located at 911 cm⁻¹, 1657 cm⁻¹, 1555 cm⁻¹, 1483 cm⁻¹, 1695 cm⁻¹, and 3309 cm⁻¹ are characteristic vibrations of calycosin (Xie et al., 2018). The PEG-PPG-PEG peaks show the characteristic vibration of PEG and PPG (Lee et al., 2008). Afterwards, some common characteristic vibrations can be observed in calycosin-PEG-PPG-PEG bands, indicating the successful preparation of the drug-loaded nanomicelles.

The CMC of the PEG-PPG-PEG copolymer was determined based on the solubilization of DPH probe after interaction with hydrophobic micellar cores and corresponding absorbance changes at 390 nm. Fig. 1D shows the changes in the absorbance of PEG-PPG-PEG/DPH micellar dispersions at different copolymer concentrations. Based on Fig. 1D, the aggregation process was significantly observed above a concentration of 0.086 mg/mL.

It was also shown that DL% and EE% of calycosin-PEG-PPG-PEG nanomicelles were 7.38 ± 1.15% and 87.61 ± 3.86%, respectively.

4.2 In vitro drug release and antioxidant activity studies

The in vitro release profile of calycosin from PEG-PPG-PEG nanomicelles was explored in a PBS buffer with pH of 7.4 (Fig. 2A). The outcome showed a sustained calycosin release in the PBS buffer with a weaker burst phase relative to free calycosin. The determined sustained calycosin release was potentially due to the hydrophobic forces between PPG micellar core and calycosin. Indeed, due to the instability of and calycosin at physiological conditions, the nano-formulation of calycosin into the nanomicelles could be a potential candidate for its stabilization. However, during the encapsulation phase, some degradation and inactivation of drugs can be induced. Therefore, the antioxidant activity of calycosin-PEG-PPG-PEG nanomicelles was explored to assess their function. Fig. 2B showed the antioxidant activity explored by ABTS radical and hypochlorous scavenging assays, respectively. As shown in Fig. 2B, the ability of the calycosin-PEG-PPG-PEG nanomicelles to neutralize the ABTS radical and hypochlorite ions was significantly greater than that of the free calycosin. Therefore, the increased antioxidant activity of calycosin-PEG-PPG-PEG nanomicelles could be associated with the improved solubility of micellar drug in comparison with that of free drug.

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Fig. 2

(A) In vitro drug release profiles of free and calycosin-PEG-PPG-PEG nanomicelles in PBS (pH 7.4). (B) Antioxidant activity of free and calycosin-PEG-PPG-PEG nanomicelles determined by the relevant experiments. Data are presented as means ± (SD) of three independent experiments.

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4.3 MTT assay

To explore whether PEG-PPG-PEG nanomicelle enhanced the bioavailability of calycosin and reduced the cytotoxicity of calcysocin in the H9C2 cells, their cytotoxic assay was done and compared (Fig. 3A). After treatment, the cells exhibited no significant reduction in viability up to the concentration of 10 µg/ml and 50 µg/ml for free calycosin and calycosin-PEG-PPG-PEG nanomicelles, respectively showing that namomicelle formulation can reduce the cytotoxic effects of calycosin at high concentrations (Fig. 3A).

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Fig. 3

(A) MTT assay of H9C2 cells incubated with different concentrations (0.01–50 µg/mL) of Dox, nanomicelles, free calycosin and calycosin-PEG-PPG-PEG nanomicelles for 24 h. (B) MTT assay of cells incubated with IC₅₀ concentration of Dox and the protective effects of co-incubation of calycosin (10 µg/mL) or calycosin-PEG-PPG-PEG nanomicelles (10 µg/mL) against Dox-induced cardiotoxicity. Data are presented as means ± (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001: relative to control group; ^#P < 0.05, ^##P < 0.01: relative to Dox-incubated cells.

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It was also observed that Dox induced cytotoxicity at extremely low concentrations starting from 0.01 µg/ml (Fig. 3A) with an IC₅₀ concentration (a concentration of Dox inhibit the viability of cells by 50%) of 0.19 µg/ml. Therefore, for further studies to induce cytotoxicity in H9C2 cells by Dox, its IC₅₀ concentration was used and for the exploring the protective effects of free or nano-formulated calycosin the optimum concentration of calycosin with no significant cytotoxic effect (10 µg/ml) was used.

It was seen that the viability of the H9C2 cells was significantly decreased (***P < 0.001; n = 3) following incubation with Dox for 24 h compared with the negative control cells (Fig. 3B). By contrast, treatment of the H9C2 cells with free calycosin (^#P < 0.05; n = 3) or calycosin-PEG-PPG-PEG nanomicelles (^##P < 0.01; n = 3) caused increased cell survival, whereas, the inhibitory effect of nano-formulated calycosin was more significant than free drug (*P < 0.05; n = 3).

4.4 Ros and RNS assays

To further explore the calycosin-nanomicelles protective effects, we investigated their effects on generation of intracellular ROS and RNS (Fig. 4). Quantitative measurements of the ROS (Fig. 4A) and RNS (Fig. 4B) indicated that Dox increased ROS and RNS generation in the H9C2 cells. The increase in the intracellular ROS and RNS was markedly inhibited with free calycosin or calycosin-PEG-PPG-PEG nanomicelles, whereas calycosin in the nano-formulated platform showed a greater protective effect (*P < 0.05) against ROS and RNS generation induced by Dox in the H9C2 cells. Also, fluorescence imaging showed the number of bright green cells as a marker of ROS production was greatly decreased after treatment of cells with calycosin-PEG-PPG-PEG nanomicelles (Fig. 4C), which is in good agreement with the quantified DCF fluorescence intensity data.

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Fig. 4

(A) ROS and (B) RNS assays of H9C2 cells incubated with IC₅₀ concentration of Dox and the protective effects of co-incubation of calycosin (10 µg/mL) or calycosin-PEG-PPG-PEG nanomicelles (10 µg/mL) against Dox-induced ROS and RNS production. (C) ROS assay determined by fluorescence microscopy (i: control, ii, Dox-incubated cell, iii: Dox-nanomicelles-incubated cells, iv: Dox-calycosin-incubated cells, v: Dox-calycosin-nanomicelles-incubated cells. Data are presented as means ± (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001: relative to control group; ^#P < 0.05, ^##P < 0.01: relative to Dox-incubated cells.

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4.5 qPCR assay

It has been well-documented that excessive ROS and RNS can stimulate apoptotic death through different signaling pathways. Therefore, we examined the relative expression of different mRNA, including ERp57, p53, Bax, and Bcl-2. It was shown that upon exposure of cells to Dox, a significant overexpression in the p53 and Bax mRNA was observed, whereas ERp57 and Bcl-2 mRNA were downregulated (***P < 0.001; n = 3) (Fig. 5A). By contrast, treatment with calycosin or calycosin-nanomicelles regulated the expression of these genes, whereas the determined productive effects were more pronounced for nano-formulated drug than free one (*P < 0.05) (Fig. 5A).

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Fig. 5

(A) qPCR assay of H9C2 cells incubated with IC₅₀ concentration of Dox and the protective effects of co-incubation of calycosin (10 µg/mL) or calycosin-PEG-PPG-PEG nanomicelles (10 µg/mL) against Dox-induced ERp57, Bcl-2/p53, Bax downregulation/upregulation. (B) qPCR assay of the ERp57 mRNA expression levels of H9C2 cells incubated with blank, NC-siRNA and ERp57-siRNA. (C) Relative expression of ERp57,p53, Bax, and Bcl-2 mRNA in the presence of Dox and ERp57 siRNA. Data are presented as means ± (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001: relative to control group; ^#P < 0.05, ^##P < 0.01: relative to Dox-incubated cells.

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To more explore the signaling pathway involved in the protective effects of calycosin-PEG-PPG-PEG nanomicelles against Dox-induced cardiotoxicity in H9C2 cells, the expression levels of the selected genes were explored in cells treated with ERp57-siRNA and different treatments. As indicated in Fig. 5B, ERp57-siRNA silencing significantly downregulated the expression of ERp57 mRNA (***P < 0.05), whereas, NC-siRNA did not significantly change the expression of this mRNA.

The effects of ERp57-siRNA silencing on Dox-induced cardiotoxicity and the protective effect of calycosin-PEG-PPG-PEG nanomicelles were examined using qPCR assay. The expression level of ERp57, p53, Bax, and Bcl-2 mRNA in the cells treated with ERp57 siRNA and Dox changed significantly (***P < 0.001) in comparison with a negative control sample (Fig. 5C). Furthermore, the ERp57 siRNA/Dox-treated cells incubated with calycosin-PEG-PPG-PEG nanomicelles did not show any regulation in the expressing level of mRNA, indicating that silencing ERp57 mRNA has inhibited the protective effect of calycosin-PEG-PPG-PEG nanomicelles.

4.6 Western blot analysis

The regulation of the selected proteins was determined in cells incubated with ERp57-siRNA, Dox and calycosin-PEG-PPG-PEG nanomicelles. As indicated in Fig. 6, when H9C2 cells were treated with Dox, protein expression was significantly changed relative to negative control cells, which was almost regulated in the presense of calycosin-nanomicelles. However, once ERp57-siRNA was applied, expression levels of protein were greatly influenced, which suggested that treatment of ERp57-siRNA outstandingly prohibited the protective effects of calycosin- nanomicelles and increased protein de-regulation induced by Dox. Indeed, the expression levels of ERp57 and Bcl-2 were reduced and those of p53 and Bax were significantly increased in response to Dox. When the cells were incubated with Dox and calycosin-nanomicelles, the expression levels of these proteins were almost regulated. However, ERp57‑siRNA silencing restored the upregulation of Bax and p53 proteins and downregulation of ERp57 and Bcl-2 proteins of cells. In genral in agreement with qPCR assay, western blot analysis indicated that co-incubation of cells with ERp57-siRNA, Dox and calycosin-PEG-PPG-PEG nanomicelles reduced the protective effect of calycosin.

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Fig. 6

Western blot analyses of ERp57, p53, Bax, and Bcl-2 proteins of H9C2 cells under different treatments. GAPDH was used as the internal standard.

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It has been shown that calycosin results in the mitigation of myocardial injury (Tsai et al., 2019; Liu et al., 2020) and recovery of heart function (Huang et al., 2020). It has been also shown that calycosin leads to cardiovascular protective effects (Li et al., 2020) mediated by estrogen receptor-α/β (Liu et al., 2016). Also, it has been reported that calycosin mitigated Dox‐stimulated cardiotoxicity by inhibiting oxidative stress and inflammatory responses though sirtuin 1 pathway (Zhai et al., 2020). Based on the unique characteristics of polyphenol compounds (unwanted side effects and low solubility at high concentration), different formulation strategies have been proposed to increase their bioavailability and pharmaceutical potency (Salehi et al., 2020; Jain et al., 2015; Conte et al., 2016). PEG and PPG polymers due to their biocompatible structures and unique physicochemical properties have been widely used for preparation of self-assembled nanomicelles for hydrophobic drug delivery (Gong et al., 2010; Öcal et al., 2014; Kim et al., 2017). In the present work, we found that nano-formulation of calycosin results in improvement of its protective effects against Dox-induced cardiotoxicity in vitro.

ERp57 as a thiol oxidoreductase of the endoplasmic reticulum is widely expressed with variable levels within different tissues. Several studies have reported the diverse well-defined activities of ERp57 in different diseases. De-expression of ERp57 has been associated with different disease from cancer to neurodegenerative disorders (Hettinghouse et al., 2018). Also, it has been reported that ERp57 can be implemented as a target for some drugs as cardioprotective agents (Cui et al., 2015).

Therefore, this paper was aimed to provide some information related to regulation of ERp57 in Dox-induced cardiotoxicity and the protective effect of calycosin-loaded nanomicelles, which highlights the potential biomedical applications of targeting ERp57.

Therefore, this paper may provide a model to express the biological effect of ERp57 in Dox-induced cardiotoxicity and the side effects and limitations reversal of Dox by calycosin either alone or in the nano-formulated platform. The data indicated that Dox treatment of H9C2 cells leads to low ERp57 expression. As a result, calycosin treatment in the both forms of free and nano-formulated species can activate the EPr57 signaling pathway, which mitigates the apoptosis process. The outcomes of this study may provide useful information to overcome the cardiotoxicity challenge in the clinical treatment of different cancers.